scholarly journals Sequence specificity in DNA binding is determined by association rather than dissociation

2021 ◽  
Author(s):  
Emil Marklund ◽  
Guanzhong Mao ◽  
Sebastian Deindl ◽  
Johan Elf
Keyword(s):  
Dna Binding ◽  
Genetic Information ◽  
Specific Binding ◽  
Theoretical Framework ◽  
Simple Equation ◽  
Target Sequence ◽  
Sequence Specificity ◽  
Target Search ◽  
Target Binding ◽  
Site Recognition

AbstractSequence-specific binding of proteins to DNA is essential for accessing genetic information. Here, we derive a simple equation for target-site recognition, which uncovers a previously unrecognized coupling between the macroscopic association and dissociation rates of the searching protein. Importantly, this relationship makes it possible to recover the relevant microscopic rates from experimentally determined macroscopic ones. We directly test the equation by observing the binding and unbinding of individual lac repressor (LacI) molecules during target search. We find that LacI dissociates from different target sequences with essentially identical microscopic dissociation rates. Instead, sequence specificity is determined by the efficiency with which the protein recognizes different targets, effectively reducing its risk of being retained on a non-target sequence. Our theoretical framework also accounts for the coupling between off-target binding and unbinding of the catalytically inactive Cas9 (dCas9), showing that the binding pathway can be obtained from macroscopic data.One Sentence SummaryAssociation and dissociation rates are anti-correlated for reactions that include a nonspecific probing step.

The combination of sequence-specific and nonspecific DNA-binding modes of transcription factor SATB1

2016 ◽  
Vol 473 (19) ◽  
pp. 3321-3339 ◽  
Author(s):  
Kazuhiko Yamasaki ◽  
Tomoko Yamasaki
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Tertiary Structure ◽  
Specific Binding ◽  
Binding Mode ◽  
Matrix Attachment Region ◽  
Titration Calorimetry ◽  
Affinity Constant ◽  
Target Sequence ◽  
Binding Modes

Transcription factor SATB1 (special AT-rich sequence binding protein 1) contains multiple DNA-binding domains (DBDs), i.e. two CUT-domain repeats (CUTr1 and CUTr2 from the N-terminus) and a homeodomain, and binds to the matrix attachment region (MAR) of DNA. Although CUTr1 and the homeodomain, but not CUTr2, are known to contribute to DNA binding, different research groups have not reached a consensus on which DBD is responsible for recognition of the target sequence in MAR, 5′-TAATA-3′. Here, we used isothermal titration calorimetry to demonstrate that CUTr1 has binding specificity to this motif, whereas the homeodomain shows affinity for a variety of DNAs without specificity. In line with nonspecific DNA-binding properties of the homeodomain, a mutation of the invariant Asn at position 51 of the homeodomain (typically in contact with the A base in a sequence-specific binding mode) did not affect the binding affinity significantly. The NMR analyses and computational modeling of the homeodomain, however, revealed the tertiary structure and DNA-binding mode that are typical of homeodomains capable of sequence-specific binding. We believe that the lack of highly conserved basic residues in the helix relevant to the base recognition loosens its fitting into the DNA groove and impairs the specific binding. The two DBDs, when fused in tandem, showed strong binding to DNA containing the 5′-TAATA-3′ motif with an affinity constant >108 M−1 and retained nonspecific binding activity. The combination of the sequence-specific and nonspecific DNA-binding modes of SATB1 should be advantageous in a search for target loci during transcriptional regulation.

scholarly journals Novel DNA Bis-intercalation by MLN944, a Potent Clinical Bisphenazine Anticancer Drug

2004 ◽  
Vol 279 (44) ◽  
pp. 46096-46103 ◽  
Author(s):  
Jixun Dai ◽  
Chandanamalie Punchihewa ◽  
Prakash Mistry ◽  
Aik Teong Ooi ◽  
Danzhou Yang
Keyword(s):  
Dna Binding ◽  
Anticancer Drug ◽  
Topoisomerase I ◽  
Specific Binding ◽  
Binding Mode ◽  
Dependent Manner ◽  
Sequence Specificity ◽  
Phase I Clinical Trials ◽  
Mobility Shift ◽  
Dna Sequence Specificity

The new bisphenazine anticancer drug MLN944 is a novel cytotoxic agent with exceptional anti-tumor activity against a range of human and murine tumor models both invitroand invivo. MLN944 has recently entered Phase I clinical trials. Despite the structural similarity with its parent monophenazine carboxamide and acridine carboxamide anticancer compounds, MLN944 appears to work by a distinct mechanism of inhibiting DNA transcription rather than the expected mechanism of topoisomerase I and II inhibition. Here we present the first NMR structure of MLN944 complexed with d(ATGCAT)2DNA duplex, demonstrating a novel binding mode in which the two phenazine rings bis-intercalate at the 5′-TpG site, with the carboxamide amino linker lying in the major groove of DNA. The MLN944 molecule adopts a significantly unexpected conformation and side chain orientation in the DNA complex, with the N10 on the phenazine ring protonated at pH 7. The phenazine chromophore of MLN944 is very well stacked with the flanking DNA base pairs using the parallel base-stacking intercalation binding mode. The DNA sequence specificity and the groove recognition of MLN944 binding is determined by several site-specific hydrogen bond interactions with the central G:C base pair as well as the favorable stacking interactions with the 5′-flanking thymine. The specific binding site of MLN944 is known to be recognized by a number of important transcription factors. Our electrophoretic gel mobility shift assay results demonstrated that the c-Jun DNA binding to the AP-1 site is significantly inhibited by MLN944 in a dose-dependent manner. Thus, the exceptional biological activity of MLN944 may be due to its novel DNA binding mode leading to a unique mechanism of action.

scholarly journals Unusual DNA-binding properties of the Arabidopsis thaliana WRKY50 transcription factor at target gene promoters

2020 ◽  
Author(s):  
Konstantin Kanofsky ◽  
Jendrik Rusche ◽  
Lea Eilert ◽  
Fabian Machens ◽  
Reinhard Hehl
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Target Gene ◽  
Target Genes ◽  
Specific Binding ◽  
Gene Promoters ◽  
Molecular Pattern ◽  
High Flexibility ◽  
Site Recognition

Abstract Key message WRKY50 from A. thaliana requires WT-boxes at target gene promoters for activation and binding. Abstract Based on the genome-wide prediction of WRKY50 target genes and the similarity of a WRKY50 binding site to WT-boxes in microbe-associated molecular pattern (MAMP)-responsive cis-regulatory modules (CRM), four WT-box containing CRMs from the promoter region of three WRKY50 target genes were investigated for their interaction with WRKY50. These target genes are DJ1E, WRKY30 and ATBBE4. Two of the four CRMs, one from DJ1E and one from WRKY30, were able to activate reporter gene expression in the presence of WRKY50. Activation requires the WT-boxes GGACTTTT, GGACTTTG from DJ1E and GGACTTTC from WRKY30. WRKY50 does not activate a second CRM from WRKY30 and the CRM from ATBBE4, both containing the WT-box TGACTTTT. In vitro gel-shift assays demonstrate WT-box-specific binding of the WRKY50 DNA-binding domain to all four CRMs. This work shows a high flexibility of WRKY50 binding site recognition beyond the classic W-box TTGACC/T.

scholarly journals CRISPR-Cas12a target binding unleashes single-stranded DNase activity

2017 ◽  
Author(s):  
Janice S. Chen ◽  
Enbo Ma ◽  
Lucas B. Harrington ◽  
Xinran Tian ◽  
Jennifer A. Doudna
Keyword(s):  
Dna Binding ◽  
Fundamental Property ◽  
Dnase Activity ◽  
Target Sequence ◽  
Sequence Matching ◽  
Genetic Changes ◽  
Guide Rna ◽  
Circular Ssdna ◽  
Target Binding ◽  
Type V

AbstractCRISPR-Cas12a (Cpf1) proteins are RNA-guided DNA targeting enzymes that bind and cut DNA as components of bacterial adaptive immune systems. Like CRISPR-Cas9, Cas12a can be used as a powerful genome editing tool based on its ability to induce genetic changes in cells at sites of double-stranded DNA (dsDNA) cuts. Here we show that RNA-guided DNA binding unleashes robust, non-specific single-stranded DNA (ssDNA) cleavage activity in Cas12a sufficient to completely degrade both linear and circular ssDNA molecules within minutes. This activity, catalyzed by the same active site responsible for site-specific dsDNA cutting, indiscriminately shreds ssDNA with rapid multiple-turnover cleavage kinetics. Activation of ssDNA cutting requires faithful recognition of a DNA target sequence matching the 20-nucleotide guide RNA sequence with specificity sufficient to distinguish between closely related viral serotypes. We find that target-dependent ssDNA degradation, not observed for CRISPR-Cas9 enzymes, is a fundamental property of type V CRISPR-Cas12 proteins, revealing a fascinating parallel with the RNA-triggered general RNase activity of the type VI CRISPR-Cas13 enzymes.One Sentence SummaryCas12a (Cpf1) and related type V CRISPR interference proteins possess non-specific, single-stranded DNase activity upon activation by guide RNA-dependent DNA binding.

scholarly journals Molecular Characterization of GrlA, a Specific Positive Regulator of ler Expression in Enteropathogenic Escherichia coli

2010 ◽  
Vol 192 (18) ◽  
pp. 4627-4642 ◽  
Author(s):  
Rafael Jiménez ◽  
Sara B. Cruz-Migoni ◽  
Alejandro Huerta-Saquero ◽  
Víctor H. Bustamante ◽  
José L. Puente
Keyword(s):  
Escherichia Coli ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Protein Misfolding ◽  
Specific Binding ◽  
Regulatory Region ◽  
Negative Regulator ◽  
Single Amino Acid ◽  
Binding Motif ◽  
Target Sequence

ABSTRACT Enteropathogenic Escherichia coli (EPEC) infections are characterized by the formation of attaching and effacing (A/E) lesions on the surfaces of infected epithelial cells. The genes required for the formation of A/E lesions are located within the locus of enterocyte effacement (LEE). Ler is the key regulatory factor controlling the expression of LEE genes. Expression of the ler gene is positively regulated by GrlA, which is encoded by the LEE. Here, we analyze the mechanism by which GrlA positively regulates ler expression and show that in the absence of H-NS, GrlA is no longer essential for ler activation, further confirming that GrlA acts in part as an H-NS antagonist on the ler promoter. Single-amino-acid mutants were constructed to test the functional significance of the putative helix-turn-helix (HTH) DNA binding motif found in the N-terminal half of GrlA, as well as at the C-terminal domain of the protein. Several mutations within the HTH motif, but not all, completely abolished GrlA activity, as well as specific binding to its target sequence downstream from position −54 in the ler regulatory region. Some of these mutants, albeit inactive, were still able to interact with the negative regulator GrlR, indicating that loss of activity was not a consequence of protein misfolding. Additional residues in the vicinity of the HTH domain, as well as at the end of the protein, were also shown to be important for GrlA activity as a transcriptional regulator, but not for its interaction with GrlR. In summary, GrlA consists of at least two functional domains, one involved in transcriptional activation and DNA binding and the other in heterodimerization with GrlR.

scholarly journals Rely on Each Other: DNA Binding Cooperativity Shapes p53 Functions in Tumor Suppression and Cancer Therapy

Cancers ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 2422
Author(s):  
Oleg Timofeev ◽  
Thorsten Stiewe
Keyword(s):  
Cancer Therapy ◽  
Dna Binding ◽  
Cell Fate ◽  
Tumor Suppression ◽  
Quaternary Structure ◽  
Three Dimensional ◽  
Structural Basis ◽  
Sequence Specificity ◽  
Cell Fate Decisions ◽  
Cancer Mutations

p53 is a tumor suppressor that is mutated in half of all cancers. The high clinical relevance has made p53 a model transcription factor for delineating general mechanisms of transcriptional regulation. p53 forms tetramers that bind DNA in a highly cooperative manner. The DNA binding cooperativity of p53 has been studied by structural and molecular biologists as well as clinical oncologists. These experiments have revealed the structural basis for cooperative DNA binding and its impact on sequence specificity and target gene spectrum. Cooperativity was found to be critical for the control of p53-mediated cell fate decisions and tumor suppression. Importantly, an estimated number of 34,000 cancer patients per year world-wide have mutations of the amino acids mediating cooperativity, and knock-in mouse models have confirmed such mutations to be tumorigenic. While p53 cancer mutations are classically subdivided into “contact” and “structural” mutations, “cooperativity” mutations form a mechanistically distinct third class that affect the quaternary structure but leave DNA contacting residues and the three-dimensional folding of the DNA-binding domain intact. In this review we discuss the concept of DNA binding cooperativity and highlight the unique nature of cooperativity mutations and their clinical implications for cancer therapy.

scholarly journals Determinants for DNA-binding site recognition by the glucocorticoid receptor.

1992 ◽  
Vol 267 (35) ◽  
pp. 24941-24947
Author(s):  
J Zilliacus ◽  
A.P. Wright ◽  
U Norinder ◽  
J.A. Gustafsson ◽  
J Carlstedt-Duke
Keyword(s):  
Dna Binding ◽  
Glucocorticoid Receptor ◽  
Binding Site ◽  
Dna Binding Site ◽  
Site Recognition

scholarly journals CRISPR as a Diagnostic Tool

Biomolecules ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 1162
Author(s):  
Seohyun Kim ◽  
Sangmin Ji ◽  
Hye Ran Koh
Keyword(s):  
Diagnostic Tool ◽  
Specific Binding ◽  
Diagnostic Methods ◽  
Specific Gene ◽  
Guide Rna ◽  
Engineering Technique ◽  
Target Dna ◽  
Target Binding ◽  
Genetic Engineering Technique ◽  
Disease Related Gene

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system has recently gained growing attention as a diagnostic tool due to its capability of specific gene targeting. It consists of Cas enzymes and a guide RNA (gRNA) that can cleave the target DNA or RNA based on the sequence of the gRNA, making it an attractive genetic engineering technique. In addition to the target-specific binding and cleavage, the trans-cleavage activity was reported for some Cas proteins, including Cas12a and Cas13a, which is to cleave the surrounding single-stranded DNA or RNA upon the target binding of Cas-gRNA complex. All these activities of the CRISPR-Cas system are based on its target-specific binding, making it applied to develop diagnostic methods by detecting the disease-related gene as well as microRNAs and the genetic variations such as single nucleotide polymorphism and DNA methylation. Moreover, it can be applied to detect the non-nucleic acids target such as proteins. In this review, we cover the various CRISPR-based diagnostic methods by focusing on the activity of the CRISPR-Cas system and the form of the target. The CRISPR-based diagnostic methods without target amplification are also introduced briefly.

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